NSUN2 facilitates DICER cleavage of DNA damage-associated R-loops to promote repair.

DNA integrity is constantly challenged by both endogenous and exogenous damaging agents, resulting in various forms of damage. Failure to repair DNA accurately leads to genomic instability, a hallmark of cancer. Distinct pathways exist to repair different types of DNA damage. Double-strand breaks (DSBs) represent a particularly severe form of damage, due to the physical separation of DNA strands. The repair of DSBs requires the activity of RNA Polymerase II (RNAPII) and the generation of Damage-responsive transcripts (DARTs). Here we show that the RNA m(5)C-methyltransferase NSUN2 localises to DSBs in a transcription-dependent manner, where it binds to and methylates DARTs. The depletion of NSUN2 results in an accumulation of nascent primary DARTs around DSBs. Furthermore, we detect an RNA-dependent interaction between NSUN2 and DICER, which is stimulated by DNA damage. NSUN2 activity promotes DICER cleavage of DARTs-associated R-loops, which is required for efficient DNA repair. We report a role of the RNA m(5)C -methyltransferase NSUN2 within the RNA-dependent DNA damage response, highlighting its function as a DICER chaperone for the clearance of non-canonical substrates such as DARTs, thereby contributing to genomic integrity.